P996: A NOVEL GERMLINE GAIN-OF-FUNCTION JAK2 MUTATION: L604F
Authors: Lukas Dvoracek, Jana Markova, Ales Holoubek, Dana Grebenova, Katerina Kuzelova, Jiří Schwarz
Journal: HemaSphere (2023)

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: The somatic V617F JAK2 mutation is found in 95% of patients with polycythemia vera (PV) and is considered the main molecular cause in a substantial part of myeloproliferative neoplasm (MPN) cases. Some rare alternative JAK2 mutations have been described. According to a recently published structural model (Glassman et al., Science 2022), the V-to-F change improves the stability of JAK2 dimer, thereby enhancing downstream signaling, e.g. via the transcription factors STAT. The impact of V617F, as well as of other activating JAK2 mutations, critically depends on the presence of F595, which is located at the interface between JAK2 monomers in the dimer. The effects of V617F may be modulated by co-occurring germline mutations, which usually does not largely alter JAK2 signaling in themselves. Aims: Functional characterization of a novel germline JAK2 mutation. Methods: JAK2 mutation screening using next-generation sequencing (NGS) was performed in 189 patients with polyglobulia or thrombocytosis with unclear molecular background. Functional consequences of the novel mutation, L604F, were compared with those induced by V617F in two different in vitro model systems: (i) HEK293T cells transfected with plasmids for exogenous JAK2-GFP expression and (ii) HeLa cells with endogenous JAK2 modifications performed using CRISPR/Cas9. Results: NGS screening revealed a previously undescribed mutation, JAK2 L604F, which was germline, homozygous, and co-occurring with V617F in an 18-y female patient with PV. Her father with heterozygous L604F had no clinical symptoms, but his blood screen results corresponded to PV. No other mutation explaining the PV phenotype was detected by NGS panel in this family member. The observed in vitro effects of L604F change were similar to those of V617F, although slightly less pronounced. In HEK293T cells, both mutated JAK2 variants significantly increased STAT5 and STAT3 phosphorylation compared to the wild-type JAK2. In contrast, HeLa cells with either L604F or V617F JAK2 modification had significantly lower JAK2 protein levels compared to the parental cells, whereas no increase in STAT phosphorylation was found. In the first system, the novel L604F mutation, but not V617F, was able to partially rescue JAK2 loss of function due to F595 change to alanine (see Figure). Summary/Conclusion: The germline L604F JAK2 mutation activates cytokine-independent signaling similarly to the acquired V617F. L604 is located between the residues F595 and V617, which form the critical part of the interface between JAK2 monomers in the active dimer. Thus, F604 is most likely involved in dimer formation. We propose that L604F mutation increases the probability of spontaneous JAK2 dimer formation mediated by F595. In addition, L604F likely contributes to dimer stabilization similarly to V617F. Financial support: Ministry of Health of the Czech Republic, project for conceptual development of the research organization 00023736.Keywords: Myeloproliferative disorder, Polycythemia vera, STAT3, STAT5